CN111315404A - Cancer treatment using pre-existing microbial immunity - Google Patents

Abstract

Methods, compositions, and kits for redirecting a pre-existing immune response in an individual to reduce or stabilize cancer in the individual.

Description

Cancer treatment using pre-existing microbial immunity

Cross reference to related applications

This application claims the benefit of U.S. provisional patent application serial No. 62/582,097, filed on 6/11/2017, which is incorporated herein by reference.

Technical Field

The present invention relates to immunology and cancer therapy, including methods, compositions and kits for directing the immune response present in a patient to cancer.

Background

In healthy individuals, persistent asymptomatic viral infection is usually controlled by cell-mediated immunity and/or humoral immunity, but can be reactivated in immunocompromised individuals cell-mediated immunity increases with age and leads to induction of many fully functional virus-specific T cells Cytomegalovirus (CMV) is a β herpes virus that is highly prevalent worldwide (infecting 50-90% of the population) and is generally asymptomatic in healthy individuals, CMV establishes lifelong persistent infections that require long-term cellular immunity to prevent disease, thus CMV reactivation is a threat in the case of immunosuppression (e.g. in hematopoietic stem cell transplantation) in immunocompetent individuals, CD4 and CD 8T cell responses to CMV exhibit broad reactivity and high intensity against multiple CMV antigens, is highly prevalent in the general population, and increases with age (m.bajwa et al, J inffect Dis 215,1212-20) T cell responses are highly prevalent in the non-oncotic population, and non-persistent memory cell responses (CMV) are considered to be of the type after persistent T cell-expansion (CMV-induced by the resting T cell-mediated immune response is a) that is a non-oncotic in humans, (CD8) type of persistent immune response after persistent infection (CD 8).

Induction of anti-tumor T cell responses is crucial in the development of effective immunotherapy against cancer. Only a fraction of cancer patients respond to current immunotherapy. Generating T cell immunity against cancer antigens often requires highly personalized approaches or relies on pre-existing anti-cancer T cells. It is also difficult to generate potent neonatal T cell immunity in cancer patients, particularly in the elderly. The personalization method relies on vaccines directed against tumor-associated antigens, neoantigens (i.e., mutated autoantigens), or viral oncoproteins. Other methods are based on adoptive transfer of chimeric antigen receptor transduced T cells or infusion of monoclonal antibodies that require laborious identification of tumor specific antigens and are applicable to only a subset of cancer types or subtypes. Finally, adoptive transfer of ex vivo expanded tumor-specific lymphocytes is a method aimed at exploiting the naturally occurring anti-tumor response. All of these methods are highly personalized and require the identification of tumor epitopes and/or ex vivo expansion of autologous cells of the patient.

In parallel, in situ tumor immunotherapies based on cytokines or TLR ligands have been used, but they mainly target innate immune recognition mechanisms to alter the tumor immune microenvironment, thereby triggering immunogenic cancer cell death and promoting epitope spreading.

Thus, there remains a need for a simple, broadly applicable, antigen-agnostic method of immunotherapy to exploit the role of the immune system in early and long-term cancer control by direct killing and promotion of epitope spreading, respectively.

Disclosure of Invention

The present inventors have recognized that sophisticated adaptive cell-mediated immunity that has developed over the years to strongly control chronic viral infection in aging humans is a type of cell-mediated immunity that is effective in controlling tumor growth. To treat cancer with this type of antiviral immunity, the inventors developed a new method of in situ immunotherapy by targeting the tumor environment directly with highly functional pre-existing antiviral T cells, either by using tumor-avid (tumor-tropic) papillomavirus pseudovirions or by injecting minimal viral CD8 and CD 4T cell Cytomegalovirus (CMV) epitopes in situ. Presentation of viral epitopes in the tumor environment results in situ recruitment and activation of viral antigen-specific T cells, leading to killing of otherwise virus-negative tumor cells and alterations in the tumor microenvironment. This approach responds to an unmet need as it meets all the criteria for successful immunotherapy by promoting and establishing both early and long-term cancer cell killing and epitope spreading.

Accordingly, the present disclosure provides methods of treating cancer in an individual by recruiting a preexisting immune response to the site of the cancer, thereby treating the cancer. The pre-existing immune response may be an immune memory response present in the individual diagnosed as pre-cancerous. The pre-existing immune response may be a naturally occurring pre-existing immune response.

In these methods, recruiting a pre-existing immune response to the cancer cells can include introducing an antigen not expressed by the cancer cells into the cancer prior to initiation of treatment, wherein the antigen is recognized by one or more components of the pre-existing immune response.

These methods may include confirming that the individual has a pre-existing immune response to the antigen prior to introducing the antigen into the tumor. These methods may also include assessing a pre-existing immune response of the individual against the antigen. In these methods, confirming the presence of the pre-existing immune response may comprise identifying a T cell response to the antigen in a sample from the individual.

In these methods, introducing the antigen can include injecting the antigen into the cancer. Additionally or alternatively, introducing the antigen can be accomplished by introducing a nucleic acid molecule encoding the antigen into the cancer. In these methods, the nucleic acid molecule may be DNA or RNA. For use with RNA, the RNA may be modified so that it is more resistant to degradation. The nucleic acid molecule can be introduced into the cancer cell by injection. Additionally or alternatively, the nucleic acid molecule may be introduced into the cancer using a viral vector or pseudovirion, such as a papillomavirus pseudovirion.

In these methods, the antigen may be a viral antigen. For example, an antigen can be a polypeptide comprising at least one epitope from a Cytomegalovirus (CMV) protein that is recognized by one or more components of a pre-existing immune response. In these methods, the CMV protein may be selected from the group consisting of: pp50, pp65, pp150, IE-1, IE-2, gB, US2, US6, UL16 and UL 18. The polypeptide may be a 9-15 mer MHC I restricted peptide. Additionally or alternatively, the polypeptide may be an MHC II restricted peptide that is at least 15-mer. Additionally or alternatively, the antigen comprises a sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID NOs 1-67. In these methods, one or more components of the immune response may be T cells.

In these methods, recruitment of a preexisting immune response can alter the microenvironment of the cancer.

In these methods, the antigen may be administered in combination with an agent that enhances the immune response. Exemplary agents include agents selected from the group consisting of: a TLR agonist; an IL-1R8 cytokine antagonist; intravenous immunoglobulin (IVIG); peptidoglycan isolated from a gram positive bacterium; lipoteichoic acid isolated from gram positive bacteria; lipoproteins isolated from gram positive bacteria; lipid arabinomannan isolated from mycobacteria, zymosan isolated from yeast cell walls; poly (a) -poly (uridylic acid); poly (IC) (poly (IC)); a lipopolysaccharide; monophosphoryl lipid a; flagellin; gadimod (Gardiquimod); imiquimod (Imiquimod); r848; an oligonucleotide containing a CpG motif, a CD40 agonist, and 23S ribosomal RNA. In an exemplary method, the antigen can be administered in combination with a poly-IC.

Another aspect provides a kit for testing a patient and recruiting a pre-existing immune response to a site of cancer in the patient. These kits can include at least one CMV peptide antigen or nucleic acid encoding a peptide, a pharmaceutically acceptable carrier, a container, and a package insert or label indicating administration of the CMV peptide for reducing at least one symptom of cancer in the patient.

This summary is neither intended nor should it be construed as being representative of the full extent and scope of the present invention. Furthermore, references herein to "the present disclosure" or various aspects thereof should be understood to refer to certain embodiments of the invention and should not necessarily be construed as limiting all embodiments to the specific description. The present disclosure is set forth in various levels of detail in this summary as well as the figures and the detailed description, and there is no intention in this summary to limit the scope of the disclosure by including or not including elements, components, etc. Other aspects of the invention will become apparent from the detailed description, particularly when taken in conjunction with the accompanying drawings.

Drawings

Figure 1A shows that murine cytomegalovirus (mCMV) infection induces a large-scale cytokine response against the mCMV peptide library (peptide pool). FIG. 1B shows IFN-gamma production by splenic CD4+ and CD8+ T cells following peptide restimulation with indicated MHC-I and MHC-II restricted mCMV peptides.

Figure 2A shows an injection protocol for intratumoral transduction of solid tumors with HPV Psv expressing the mCMV antigen. FIGS. 2B and 2C show tumor volumes following intratumoral injection of HPV16 Psv expressing m122 and m45 or HPV Psv expressing Red Fluorescent Protein (RFP), respectively.

Figure 3A depicts an injection protocol for intratumoral transduction of solid tumors with mCMV antigen expressing HPV Psv in combination with poly (I: C) (PIC). FIGS. 3B-3E show that this intratumoral transduction regimen slows tumor growth. Figures 3F and 3G show tumor infiltration by E7, m45, and m 122-specific CD8+ T cells by MHC-I tetramer staining and FACS analysis.

FIG. 4A shows the effect on survival, and FIG. 4B shows the effect on tumor growth following intratumoral injection of the MCMV MHC-I restricted peptide in C57Bl/6 mice infected with murine cytomegalovirus (mCMV).

FIG. 5 shows the effect of different doses of mCMV MHC-I restricted peptide intratumoral injection on tumor growth in C57Bl/6 mice infected with murine cytomegalovirus (mCMV).

FIGS. 6A and 6B show the effect of intratumoral injection of a combination of mCMV MHC-I and MHC-II restricted peptides on tumor growth in C57Bl/6 mice infected with mCMV. Figure 6C shows E7, m45, m122 specific CD8+ T cell responses in blood as analyzed by FACS using MHC-I tetramers for each peptide, demonstrating that sequential intratumoral vaccination with mCMV CD4 followed by CD8 epitope preferentially induces anti-tumor immunity.

Figure 7 shows the effect of complete clearance of primary tumors on long-term protection against secondary tumor challenge.

Figure 8 shows that mCMV infection induced a swelling CD8+ T cell response in C57Bl/6 mice.

FIG. 9A shows that both swelling and non-swelling CD8+ T cells produce IFN- γ and CD4+ T cells produce IFN- γ. Figure 9B shows cytokine production by mCMV CD8+ T cells against the MHC-I restricted peptide library.

Figure 10A shows the experimental protocol timing for the mouse TC1 tumor model for intratumoral administration of mCMV peptides. Fig. 10B and 10C show the distribution of mCMV-specific CD8+ T cells in tumor-bearing mice. Both swelling (IE 3; FIG. 10B) and non-swelling (m 45; FIG. 10C) specific CD8+ T cells were examined by FACS using MHC-I tetramer staining.

Fig. 11A shows experimental protocol timing for a mouse TC1 tumor model for gene expression analysis of tumor microenvironment. Fig. 11B-11F show tumor infiltration of CD45+ cells (fig. 11B), Th1 cells (fig. 11C), cytotoxic CD 8T cells (fig. 11D), NK cells (fig. 11E), or dendritic cells (fig. 11F) following intratumoral therapy.

Figures 12A and 12B show that intratumoral injection of the mCMV CD8 epitope delayed tumor growth. Co-injection of poly (I: C) improves tumor control. FIG. 12A shows the effect of intratumoral injection of MHC-I restricted mCMV peptide +/-poly (I: C) alone. Figure 12B shows the effect of intratumoral injection of MHC-I restricted mCMV peptide titration.

FIGS. 13A and 13B show that protection from TC1 tumor challenge is provided by intratumoral injection of mCMV MHC-I and/or MHC-II peptides with poly (I: C). Sequential intratumoral vaccination with CD4, followed by CD8 MCMV epitopes inhibited tumor growth (fig. 13A) and promoted long-term survival (fig. 13B).

Figure 14 shows E7 tetramer positive CD8+ T cell responses in blood after 6 treatments with MHC-I restricted selected m38, m45 and m122 peptides and/or MHC-II restricted m139 selected peptides with or without poly (I: C) (30ug) and saline or poly (I: C) alone as a control.

Figure 15 shows that complete clearance of the primary tumor confers long-term protection against secondary tumor challenge.

FIG. 16 shows that protection from MC38 tumor challenge is provided by intratumoral injection of mCMV MHC-I and MHC-II peptides with poly (I: C).

Detailed Description

The present invention relates to novel methods of treating cancer. In particular, the present invention relates to methods of treating cancer in an individual using the individual's own immune system to attack cancer cells. The method makes use of the fact that: the individual has a pre-existing immune response that is not initially elicited in response to cancer, but is instead elicited by microorganisms in the environment. Since cancer cells do not typically express microbial antigens that elicit a pre-existing immune response, it cannot be expected that such an immune response will attack the cancer. However, the inventors have found that such a pre-existing immune response can be recruited to attack cancer. One way in which this can be achieved is by introducing one or more antigens recognized by a pre-existing immune response into the cancer, thereby causing the cells of the immune response to attack the cancer cells displaying the antigen. Thus, these methods are not directed to cancer cells that express an antigen prior to treatment of a cancer patient. For example, many glioblastoma cancer cells are found to express CMV antigens, and the methods of the present disclosure would not be used to treat such glioblastoma with pre-existing immunization of individuals against CMV. In addition, destruction of cancer cells can result in exposure of components of the pre-existing immune response to cancer cell antigens. This can result in the initiation of an immune response against the cancer cell antigen. Thus, the general methods of the invention can be practiced by recruiting a pre-existing immune response in an individual to the site of cancer, such that the pre-existing immune response attacks the cancer. For example, recruitment can be achieved by introducing into the cancer at least one antigen recognized by a component of the pre-existing immune response of the individual (e.g., T cells).

The present invention is not limited to the particular embodiments described herein, as they may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. Thus, the terms "a", "an", "one or more" and "at least one" may be used interchangeably. Similarly, the terms "comprising," "including," and "having" are used interchangeably. It should also be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," etc. or use of a "negative" limitation with respect to the recitation of claim elements.

Certain features of the invention that are described in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments are expressly contemplated by the present invention and are disclosed herein as if each combination were individually and specifically disclosed. In addition, all sub-combinations are also expressly contemplated by the present invention and are disclosed herein as if each such sub-combination were individually and expressly disclosed herein.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

One aspect is a method of treating cancer in an individual comprising recruiting a preexisting immune response to the cancer, thereby treating the cancer.

As used herein, cancer refers to a disease in which abnormal cell division does not properly control cell division and/or cell senescence. The term cancer is intended to encompass solid tumors as well as blood-borne cancers. Typically, a tumor is an abnormal tissue mass that generally does not contain cysts or fluid areas. Solid tumors can be benign (not life-threatening) or malignant (life-threatening). Different types of solid tumors are named for the cell types that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. Hematological cancers (also known as hematological cancers) are cancers that begin in blood-forming tissues (such as bone marrow) or cells of the immune system. Examples of hematological cancers include leukemia, lymphoma, and multiple myeloma.

In some cancers, cells may invade tissues other than the tissue from which the original cancer cell originated. In some cancers, cancer cells can spread to other parts of the body through the blood and lymphatic system. Thus, cancer is often named after the organ or cell type from which it begins. For example, cancers that originate in the colon are referred to as colon cancers; cancers that originate in melanocytes of the skin are called melanoma and the like. As used herein, cancer may refer to carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, and the like, including solid and lymphoid cancers, gastric (gastic) cancers, renal cancers, breast cancers, lung cancers (including non-small cell and small cell lung cancers), bladder cancers, colon cancers, ovarian cancers, prostate cancers, pancreatic cancers, gastric (stomach) cancers, brain cancers, head and neck cancers, skin cancers, uterine cancers, testicular cancers, esophageal cancers, liver cancers (including liver cancers), lymphomas including non-hodgkin lymphomas (e.g., burkitt's, small cell and large cell lymphomas) and hodgkin lymphomas, leukemias and multiple myelomas. In exemplary embodiments, the cancer is lung cancer or adenocarcinoma.

As used herein, the terms individual, subject, patient, etc. are intended to encompass any mammal capable of developing cancer, wherein the preferred mammal is a human. The terms individual, subject and patient do not by themselves denote a particular age, sex, race, etc. Accordingly, the present disclosure is intended to encompass individuals of any age, whether male or female. Likewise, the methods of the present invention can be applied to any human race, including, for example, caucasian (whiter), african american (black), american native, hawaii native, hispanic, asian, and european. Such features may be important. In such cases, important characteristics (e.g., age, gender, race, etc.) will be indicated. These terms also encompass both human and non-human animals. Suitable non-human animals for testing or treating cancer include, but are not limited to, companion animals (i.e., pets), food animals, work animals, or zoo animals.

As used herein, an immune or immunological response refers to the presence of a humoral and/or cellular response to one or more antigens in an individual. For purposes of this disclosure, "humoral response" refers to an immune response mediated by B cells and antibody molecules, including secretory (IgA) or IgG molecules, while "cellular response" is an immune response mediated by T lymphocytes and/or other leukocytes. An important aspect of cellular immunity relates to the antigen-specific response of cytolytic T Cells (CTLs). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by Major Histocompatibility Complex (MHC) on the cell surface. CTLs help to induce and promote destruction of intracellular microorganisms, or lyse cells infected with such microorganisms. Another aspect of cellular immunity relates to the antigen-specific response of helper T cells. Helper T cells function to help stimulate the function of and focus on the activity of non-specific effector cells directed against cells displaying on their surface peptide antigens associated with MHC molecules. Cellular immune responses also refer to the production of cytokines, chemokines and other such molecules produced by activated T cells and/or other leukocytes, including those derived from CD4+ and CD8+ T cells.

Thus, the immunological response may be a response that stimulates the production or activation of CTLs, and/or helper T cells. Production of chemokines and/or cytokines may also be stimulated. The immune response may also comprise an antibody-mediated immune response. Thus, the immunological response may include one or more of the following effects: b cells produce antibodies (e.g., IgA or IgG); and/or the activation of inhibitory, cytotoxic or helper T cells and/or T cells specific for an antigen. Such responses can be determined using standard immunoassays and neutralization assays known in the art.

As used herein, a pre-existing immune response is an immune response that is present in an individual prior to the initiation of cancer treatment. Thus, prior to initiation of treatment for cancer with an antigen, individuals with a pre-existing immune response have an immune response against the antigen. The pre-existing immune response may be a naturally occurring immune response, or it may be an induced immune response. As used herein, a naturally-occurring pre-existing immune response is an immune response in an individual in response to an antigen (e.g., a bacterial or viral antigen) with which the individual has been inadvertently exposed. That is, individuals with a preexisting immune response are not intentionally exposed to an antigen to generate an immune response against the antigen. The pre-existing immune response induced is an immune response due to deliberate exposure to an antigen (e.g. upon receiving a vaccine). The pre-existing immune response may be a naturally occurring immune response, or the pre-existing immune response may be an induced immune response.

As used herein, the phrase "recruiting an immune response" refers to a process in which an antigen is administered to an individual such that components of the pre-existing immune response travel through the body to the location where the antigen is administered, resulting in the attack of cells displaying the antigen by components of the immune system. As used herein, a "component of an immune response" refers to a cell that can bind to an antigen and initiate an immune response against the antigen. Antigens that can be used in the practice of the present invention are any molecules that can be recognized by cells of the pre-existing immune system, particularly T cells. An example of such a compound is a protein, such as a bacterial or viral protein.

As used herein, the phrase "treating cancer" refers to various outcomes with respect to cancer. Treating cancer includes reducing the rate of increase in the number of cancer cells in the treated individual. Such a decrease in the rate of increase may be due to a slowing of cancer cell replication. Alternatively, the rate of replication of cancer cells may be unaffected and an increased number of cancer cells may be killed by the preexisting immune response. In certain aspects, treating cancer refers to a condition in which the number of cancer cells stops increasing but remains at a constant level. Such a condition may arise from the inhibition of cancer cell replication by recruitment of a preexisting immune response, or it may be due to the rate of cancer cell killing by the recruited preexisting immune response balancing the rate of production of new cancer cells. Treating cancer refers to stabilizing the cancer such that the growth of the cancer is reduced or stopped, or reducing the number of cancer cells in a treated individual and/or an individual without cancer (i.e., no cancer cells detectable).

In embodiments, the step of recruiting the pre-existing immune response comprises introducing into the cancer an antigen recognized by one or more components of the pre-existing immune response. In a preferred embodiment, the antigen is not present in the cancer prior to treatment. Accordingly, one embodiment is a method of treating cancer in an individual comprising recruiting a preexisting immune response to the cancer by introducing into the cancer an antigen recognized by one or more components of the preexisting immune response, wherein the antigen was not present in the cancer prior to treating the cancer. Thus, as described above, the pre-existing immune response may be a naturally occurring immune response or an induced immune response. Antigens can be introduced into the cancer using methods known in the art and can vary depending on the type of cancer being treated. For example, one type of cancer is a solid tumor. In such cancers, the cancer cells replicate and remain adjacent to their parent cancer cells, resulting in the formation of tissue masses formed by the adjacent cancer cells. Since such cancers are cell masses, the antigen can be delivered directly to or into the mass. One embodiment is a method of treating cancer in an individual, wherein the cancer is a solid tumor, comprising recruiting a preexisting immune response to the solid tumor by introducing into the solid tumor an antigen recognized by one or more components of the preexisting immune response, wherein the antigen was not present in the solid tumor prior to treatment. In one embodiment, the pre-existing immune response is a naturally occurring immune response. In one embodiment, the pre-existing immune response is an induced immune response. In one embodiment, the antigen is delivered to the cancer (e.g., a solid tumor) by injecting the antigen into the cancer (e.g., a solid tumor). In such embodiments, the antigen is delivered directly into the cancer, either by direct binding to such molecules or by uptake and processing of the antigen by the cancer cells, thereby allowing the antigen to be displayed on the MHC I molecule of the cell. In these methods, the antigen may be combined with other molecules or compounds that enhance the uptake of the antigen and/or presentation of the antigen to the immune system.

As previously mentioned, in these methods, the antigen may be a protein. As described above, these protein antigens can be injected directly into a cancer (e.g., a tumor). Accordingly, one embodiment is a method of treating cancer in an individual, wherein the cancer is a solid tumor, comprising recruiting a preexisting immune response to the solid tumor by injecting the solid tumor with an antigenic protein, wherein the antigenic protein is recognized by one or more components of the preexisting immune response, and wherein the antigenic protein is not present in the solid tumor prior to treatment. Alternatively, the protein antigen may be introduced into the cancer by introducing a nucleic acid molecule encoding the protein into the cancer. Accordingly, one embodiment is a method of treating cancer in an individual, wherein the cancer is a solid tumor, comprising recruiting a preexisting immune response to the solid tumor by introducing a nucleic acid molecule encoding an antigenic protein into the solid tumor, wherein the antigenic protein is recognized by one or more components of the preexisting immune response, and wherein the antigenic protein was not present in the solid tumor prior to treatment. The nucleic acid molecule encoding the antigen may be introduced into the cancer using any suitable method known in the art. One embodiment is a method of treating cancer in an individual, wherein the cancer is a solid tumor, comprising recruiting a preexisting immune response to the solid tumor by injecting a nucleic acid molecule encoding an antigenic protein into the solid tumor, wherein the antigenic protein is recognized by one or more components of the preexisting immune response, and wherein the antigenic protein was not present in the solid tumor prior to treatment. In these methods, the nucleic acid molecule encoding the antigen can be injected as a naked nucleic acid molecule (i.e., a nucleic acid molecule that is not complexed with other molecules intended to enhance delivery of the stability of the nucleic acid molecule) or the injected nucleic acid molecule encoding the antigen can be complexed with one or more compounds intended to enhance delivery, stability, or longevity of the nucleic acid molecule. Examples of such compounds include lipids, proteins, carbohydrates, and polymers, including synthetic polymers.

Nucleic acid molecules encoding more than one antigen can also be introduced into cancer using delivery vehicles such as recombinant viruses or pseudoviruses (pseudovirions). Examples of viruses that can be used to practice the methods of the invention include, but are not limited to, adenovirus, adeno-associated virus, herpes virus, and papilloma virus. The use of such viruses for delivering nucleic acid molecules is known to those skilled in the art and is also disclosed in U.S. patent No. 8,394,411, which is incorporated herein by reference. Examples of pseudoviruses that can be used to practice the methods of the invention include, but are not limited to, hepatitis pseudoviruses, influenza pseudoviruses, and papilloma pseudoviruses. As used herein, pseudovirus refers to a particle comprising viral capsid proteins assembled into virus-like particles (VLPs) that are capable of binding to and entering cancer cells. Such pseudovirions can, but preferably do not, package subgenomic amounts of viral nucleic acid molecules. Methods of producing and using pseudovirions are known in the art and are also described in U.S. patent nos. 6,599,739; 7,205,126, respectively; and 6,416,945, which are all incorporated herein by reference in their entirety. Accordingly, the present disclosure provides a method of treating cancer in an individual, wherein the cancer is a solid tumor, comprising recruiting a preexisting immune response to the solid tumor by introducing into the tumor a recombinant or pseudovirus comprising a nucleic acid molecule encoding an antigenic protein, wherein the antigenic protein is recognized by one or more components of the preexisting immune response, and wherein the antigenic protein was not present in the solid tumor prior to treatment. Entry of a pseudovirus carrying a nucleic acid molecule of the present disclosure into a cell causes the cell to express the encoded antigenic protein and subsequently display the antigen to the immune system. In these methods, the pseudovirus is a papilloma pseudovirus.

Introduction of a virus or pseudovirus comprising a nucleic acid molecule encoding an antigen into a cancer may be achieved using any suitable method known in the art. For example, a recombinant virus or pseudovirus comprising a nucleic acid molecule encoding an antigen can be injected in the vicinity of a cancer or directly into a cancer. Alternatively, a recombinant virus or pseudovirus comprising a nucleic acid molecule encoding an antigen can be administered to an individual by a route that results in delivery of the recombinant virus or pseudovirus to a cancer. Examples of such routes include, but are not limited to, Intravenous (IV) injection, Intramuscular (IM) injection, Intraperitoneal (IP) injection, Subcutaneous (SC) injection, and oral delivery. Accordingly, one embodiment is a method of treating cancer in an individual comprising administering to the individual a recombinant or pseudovirus comprising a nucleic acid molecule encoding an antigenic protein, wherein the cancer is a solid tumor, wherein the antigenic protein is recognized by one or more components of a preexisting immune response, and wherein the antigenic protein is not present in the solid tumor prior to treatment. In these methods, the recombinant virus or pseudovirus may be directly injected into the solid tumor, or may be delivered using a method selected from the group consisting of IV injection, IM injection, IP injection, SC injection, and oral delivery.

The methods of the present disclosure may be used to treat blood-borne cancers. Blood-borne cancers, hematological cancers, and the like begin in blood-forming tissues (e.g., bone marrow) or cells of the immune system. Examples of hematological cancers include leukemia, lymphoma, and multiple myeloma. Such cancers begin when the cells of the blood-forming tissue or cells of the immune system lose control of cell replication and begin to replicate in an uncontrolled manner. Once formed, blood cancer cells can enter the blood or lymphatic system, resulting in a significant increase in the number of cancer cells in the blood and/or lymphatic system. For example, leukemia is a cancer found in the blood and bone marrow. Leukemia is caused by uncontrolled replication of leukocytes, resulting in a large increase in the number of abnormal leukocytes in blood and lymphoid tissues. These abnormal leukocytes do not function properly and thus individuals with leukemia are unable to fight the infection. Accordingly, the present disclosure provides a method of treating a hematologic cancer in an individual comprising recruiting a preexisting immune response to a hematologic cancer cell in the individual by introducing into the hematologic cancer cell an antigen recognized by one or more components of the preexisting immune response, wherein the antigen was not present in, or on, the hematologic cancer cell prior to treatment. In these methods, the pre-existing immune response may be a naturally occurring immune response or an induced immune response. Introduction of the antigen into the hematologic cancer cells can be performed using any suitable method. In these methods, the antigen can be introduced into the hematologic cancer cells by administering the antigen to the individual in a form that results in delivery of the antigen to the hematologic cancer cells. For example, the antigen may be administered to the individual using a method selected from IV injection, IM injection, IP injection, SC injection, and oral administration. In these methods, the antigen can be targeted to the hematologic cancer cell, for example, by conjugating the antigen to a protein that binds a molecule on the hematologic cancer cell.

Antigens can also be introduced into hematologic cancer cells by introducing nucleic acid molecules encoding antigenic proteins into the hematologic cancer cells in an individual. Accordingly, the present disclosure provides a method of treating a hematologic cancer in an individual comprising recruiting a preexisting immune response to the hematologic cancer by administering to the individual a nucleic acid molecule encoding an antigenic protein, wherein the antigenic protein is recognized by one or more components of the preexisting immune response, and wherein the antigenic protein is not present in, or on, the hematologic cancer cells prior to treatment. The nucleic acid molecule encoding the antigen may be administered to the individual using any suitable method known in the art. For example, a nucleic acid molecule encoding an antigen can be injected as a naked nucleic acid molecule. Alternatively or additionally, the nucleic acid molecule encoding the antigen may be complexed with one or more compounds intended to enhance delivery, stability or longevity of the nucleic acid molecule. Examples of such compounds include lipids, proteins, carbohydrates, and polymers, including synthetic polymers.

Nucleic acid molecules encoding more than one antigen can also be introduced into hematologic cancer cells using a delivery vehicle such as a recombinant virus or pseudovirus. Examples of such delivery vehicles have been described previously herein. Examples of viruses that can be used to practice the methods of the invention include, but are not limited to, adenovirus, adeno-associated virus, herpes virus, and papilloma virus. Examples of pseudoviruses that can be used to practice the methods of the invention include, but are not limited to, hepatitis pseudoviruses, influenza pseudoviruses, and papilloma pseudoviruses. Accordingly, the present disclosure provides a method of treating a hematologic cancer in an individual comprising recruiting a preexisting immune response to a solid tumor by introducing into the tumor a recombinant or pseudovirus comprising a nucleic acid molecule encoding an antigenic protein, wherein the antigenic protein is recognized by one or more components of the preexisting immune response, and wherein the antigenic protein is not present in, or on, a hematologic cancer cell prior to treatment.

Introduction of a virus or pseudovirus comprising a nucleic acid molecule encoding an antigen into a cancer may be achieved using any suitable method known in the art. For example, a recombinant virus or pseudovirus comprising a nucleic acid molecule encoding an antigen can be administered to an individual by a route that results in delivery of the recombinant virus or pseudovirus to a cancer. Examples of such routes include, but are not limited to, Intravenous (IV) injection, Intramuscular (IM) injection, Intraperitoneal (IP) injection, Subcutaneous (SC) injection, and oral administration. Accordingly, the present disclosure provides a method of treating a hematologic cancer in an individual comprising administering to the individual a recombinant or pseudovirus comprising a nucleic acid molecule encoding an antigenic protein, wherein the antigenic protein is recognized by one or more components of a preexisting immune response, and wherein the antigenic protein is not present in, or on, a hematologic cancer cell prior to treatment. The recombinant virus or pseudovirus may be delivered using a method selected from the group consisting of: IV injection, IM injection, IP injection, SC injection, and oral administration.

The methods disclosed herein use one or more antigens to recruit a pre-existing immune response to a cancer. Any antigen can be used so long as the antigen is recognized by one or more components of the pre-existing immune response and the antigen is not present in or on the cancer cells prior to treatment. Examples of useful antigens include, but are not limited to, viral and bacterial antigens. One example of a viral antigen that can be used to practice the methods of the invention is an antigen that comprises at least one epitope from a cytomegalovirus protein. As used herein, an epitope is a cluster of amino acid residues that are recognized by the immune system, thereby eliciting an immune response. Such epitopes may consist of contiguous amino acid residues (i.e., amino acid residues that are adjacent to each other in a protein), or they may consist of amino acid residues that are non-contiguous (i.e., amino acid residues that are not adjacent to each other in a protein), but are in close, specific proximity in the final folded protein. It is generally understood by those skilled in the art that epitopes require a minimum of six amino acid residues to be recognized by the immune system. Thus, the methods of the invention may comprise the use of an antigen comprising at least one epitope from a cytomegalovirus protein. Any suitable CMV protein can be used to generate an antigen that can be used to practice the methods of the invention, so long as the antigen recruits a preexisting immune response to the cancer. Examples of CMV proteins suitable for use in the methods disclosed herein include, but are not limited to, CMV pp50, CMV pp65, CMV pp150, CMV IE-1, CMV IE-2, CMV gB, CMV US2, CMVUL16, and CMV UL 18. Examples of such proteins and useful fragments thereof are disclosed in U.S. patent publication nos. 2005/00193344 and 2010/0183647, both of which are incorporated by reference herein in their entirety. Useful fragments can also include any one or combination of peptides comprising the amino acid sequence of SEQ ID NOs: 1-67.

The disclosed methods can also be practiced using one or more antigens, each independently comprising an amino acid sequence that is a variant of at least 8 contiguous amino acid sequences from a CMV protein. As used herein, a variant refers to a protein or nucleic acid molecule that is similar in sequence to, but not identical to, a reference sequence, wherein the activity (e.g., immunogenicity) of the variant protein (or the protein encoded by the variant nucleic acid molecule) is not significantly altered. These sequence variations may be naturally occurring variations, or they may be engineered using genetic engineering techniques known to those skilled in the art. Examples of such techniques can be found in Sambrook J, Fritsch E F, Maniatis T et al, in Molecular Cloning-A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press,1989, pages 9.31-9.57 or Currentprotocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

With respect to variants, any type of alteration of the amino acid sequence is permissible so long as the resulting variant protein retains the ability to elicit an immune response. Examples of such variations include, but are not limited to, deletions, insertions, substitutions, and combinations thereof. For example, for proteins, it is well known to those skilled in the art that one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids can generally be removed from the amino and/or carboxy terminus of a protein without significantly affecting the activity of the protein. Similarly, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids can typically be inserted into a protein without significantly affecting the activity of the protein.

As noted, a variant protein may contain amino acid substitutions relative to a reference protein (e.g., a wild-type protein). Any amino acid substitution is permissible as long as the activity of the protein is not significantly affected. In this regard, it is understood in the art that amino acids can be classified based on their physical properties. Examples of such groups include, but are not limited to, charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids. Preferred variants containing substitutions are those in which an amino acid is substituted with an amino acid from the same group. Such substitutions are referred to as conservative substitutions.

Naturally occurring residues can be classified based on common side chain properties: 1) hydrophobicity: met, Ala, Val, Leu, Ile; 2) neutral hydrophilicity: cys, Ser, Thr; 3) acidity: asp and Glu; 4) alkalinity: asn, Gln, His, Lys, Arg; 5) residues that influence chain orientation: gly, Pro; and 6) fragrance: trp, Tyr, Phe.

For example, a non-conservative substitution may involve exchanging a member of one of these classes for a member from another class.

In making amino acid changes, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. The hydropathic index is: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydrophilic amino acid index in conferring interactive biological functions on proteins is generally understood in the art (Kyte et al, 1982, J.mol.biol.157: 105-31). It is known that other amino acids having similar hydropathic indices or scores may be substituted with certain amino acids and still retain similar biological activity. When the change is made based on the hydropathic index, the substitution of amino acids having a hydropathic index within. + -.2 is preferable, the substitution of amino acids within. + -. 1 is particularly preferable, and the substitution of amino acids within. + -. 0.5 is even more particularly preferable.

It is also understood in the art that substitution of like amino acids can be made effectively based on hydrophilicity, particularly where the resulting biologically functionally equivalent protein or peptide is intended for use in immunological inventions, as in the present case. The greatest local average hydrophilicity of a protein (governed by the hydrophilicity of its adjacent amino acids) is associated with its immunogenicity and antigenicity, i.e., with the biological properties of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When changes are made based on similar hydrophilicity values, substitutions of amino acids with hydrophilicity values within ± 2 are preferred, substitutions of amino acids within ± 1 are particularly preferred, and substitutions of amino acids within ± 0.5 are even more particularly preferred. Epitopes can also be identified from primary amino acid sequences based on hydrophilicity.

Desired amino acid substitutions (whether conservative or non-conservative) may be determined by one of skill in the art when such substitutions are desired. For example, amino acid substitutions may be used to identify important residues of a protein, or to increase or decrease the immunogenicity, solubility or stability of a protein. Exemplary amino acid substitutions are shown in the following table:

as used herein, the phrase "significantly affects protein activity" refers to a reduction in protein activity of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. With respect to the present invention, such activity can be measured, for example, as the ability of the protein to elicit a neutralizing antibody or elicit a T cell response. Methods for determining such activity are known to those skilled in the art.

The methods of the present disclosure may use one or more antigens that each independently comprise at least 6 contiguous amino acids, at least 10 contiguous amino acids, at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, or at least 100 contiguous amino acids from a CMV protein. The methods of the present disclosure may use one or more antigens that each independently comprise an amino acid sequence that is at least 85% identical, at least 95% identical, at least 97% identical, or at least 99% identical to at least 10 consecutive amino acids, at least 20 consecutive amino acids, at least 30 consecutive amino acids, at least 50 consecutive amino acids, at least 75 consecutive amino acids, or at least 100 consecutive amino acids from a CMV protein. The methods of the present disclosure may use one or more antigens that each independently comprise at least 6 contiguous amino acids, at least 10 contiguous amino acids, at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, or at least 100 contiguous amino acids from a CMV protein. The methods of the present disclosure may use one or more antigens each independently comprising an amino acid sequence that is at least 95% identical, at least 97% identical, or at least 99% identical to 9 to 15 consecutive amino acid residues from a CMV protein, wherein the antigen is an MHC I-restricted antigen. The methods of the present disclosure may use one or more antigens, each independently comprising 9 to 15 contiguous amino acid residues from a CMV protein, wherein the antigen is an MHC I-restricted antigen. The methods of the present disclosure may use one or more antigens comprising an amino acid sequence that is at least 95% identical, at least 97% identical, or at least 99% identical to at least 15 consecutive amino acid residues from a CMV protein, wherein the antigen is an MHC II restricted antigen. The methods of the present disclosure may use one or more antigens comprising at least 15 contiguous amino acid residues from a CMV protein, wherein the antigen is an MHC II restricted antigen. The methods of the present disclosure may use one or more antigens comprising an amino acid sequence that is at least 95% identical, at least 97% identical, or at least 99% identical to a peptide consisting of a sequence selected from the group consisting of seq id nos: a peptide comprising the amino acid sequence of SEQ ID NO 1-67 or any combination thereof. The methods of the present disclosure may use one or more antigens consisting of an amino acid sequence that is at least 95% identical, at least 97% identical, or at least 99% identical to a sequence selected from the group consisting of: a peptide comprising the amino acid sequence of SEQ ID NO 1-67 or any combination thereof. The methods of the present disclosure may use one or more antigens consisting of a sequence selected from the group consisting of: a peptide comprising the amino acid sequence of SEQ ID NO 1-67 or any combination thereof.

The methods of the invention include treating an individual for cancer by recruiting a preexisting immune response to the cancer. In these methods, it may be known that an individual has a pre-existing immune response against an antigen prior to the initiation of cancer therapy. Prior to initiating cancer therapy, individuals may be tested to confirm the presence of a pre-existing immune response. Thus, the methods can include treating cancer in an individual by confirming that the individual has a pre-existing immune response to an antigen, wherein the antigen is not present in or on the cancer. The antigen is then administered to an individual identified as having preexisting immunity, such that the antigen is introduced into the cancer, thereby treating the cancer.

Such methods may be used to treat any cancer that has been described herein, including any solid tumor and/or hematologic cancer.

Any method of confirming that the individual to be treated has a pre-existing immune response to an antigen can be used to practice the methods of the invention. Examples of such methods include identifying in a sample from an individual B cells that recognize a particular antigen, antibodies that recognize a particular antigen, T cells that recognize a particular antigen or T cell activity initiated in response to a particular antigen. Any suitable sample from an individual may be used to identify the pre-existing immune response. Examples of suitable samples include, but are not limited to, whole blood, serum, plasma, and tissue samples. As used herein, recognition of a particular antigen by a B cell, T cell, or antibody refers to the ability of such B cell, T cell, or antibody to specifically bind to the antigen. Specific binding of a B cell, T cell, or antibody to an antigen means that the B cell, T cell, or antibody binds to the specific antigen with an affinity that is greater than the binding affinity of the same B cell, T cell, or antibody to a molecule unrelated to the antigen. For example, a B cell, T cell, or antibody that recognizes an antigen from the CMV pp50 protein or that is specific for an antigen from the CMV pp50 protein binds to the CMV pp50 antigen with an affinity that is significantly greater than the binding affinity of the same B cell, T cell, or antibody for a protein not associated with the CMV pp50 protein (e.g., human albumin). Specific binding between two entities can be scientifically characterized by their dissociation constant, which is generally less than about 10^-6Less than about 10^-7Or less than about 10^-8And M. The concept of specific binding between molecules and between cells and molecules and methods of measuring such binding are well known to those of ordinary skill in the art and include, but are not limited to, enzyme immunoassays (e.g., ELISA), immunoprecipitations, immunoblot assays, and other immunoassays, such as, for example, Sambrook et al, supra, and Harlow et al, AntibodiesA Laboratory Manual (Cold Spring Harbor Labs Press, 1988). Such methods are also described in U.S. patent No. 7,172,873, which is incorporated herein by reference. Methods of measuring T cell activation in a sample from an individual are also known to those of skill in the art. Examples of such methods are disclosed in U.S. patent publication No. 2003/003485 and U.S. patent No. 5,750,356, both of which are incorporated herein by reference.

Such methods generally include contacting a T cell-containing sample from an individual with an antigen and measuring T cell activation of the sample. Methods of measuring T cell activation are also well known in the art and are also disclosed in Walker, s, et, translational infection Disease,2007:9: 165-70; and Kotton, C.N.et al. (2013) Transplantation 96,333.

Commercially available CMV test (QuantiFERON)^TMCMV, QIAGEN Sciences inc, Germantown, MD) can be used as an in vitro diagnostic test, which uses a peptide mixture mimicking human cytomegalovirus protein (CMV) to stimulate cells in heparinized whole blood. Individuals exposed to disease/infection have specific T cell lymphocytes in their blood that maintain immunological memory against the antigen (immunoreactive molecule) of the disease/infection being triggered. The addition of antigen to blood collected from primed individuals results in rapid restimulation of antigen-specific effector T cells, resulting in the release of cytokines (e.g., IFN- γ). Effector T cells are able to respond rapidly when exposed to a priming antigen. Thus, the production of IFN- γ in response to antigen exposure is a specific marker of the cellular immune response to that antigen. The IFN- γ response can be used to quantify the immune response. Detection of interferon-gamma (IFN- γ) by enzyme-linked immunosorbent assay (ELISA) was used to identify in vitro responses against peptide antigens associated with CMV infection. QuantiFERON^TMThe intended use of CMV is to monitor the level of immunity against CMV in humans.

Thus, in any method of the present disclosure for treating cancer in an individual, it may first be confirmed that the individual has a pre-existing immune response against an antigen not present in or on the cancer. Such a pre-existing immune response may be confirmed by identifying in a sample from the individual:

i) b cells recognizing a specific antigen;

ii) an antibody recognizing a specific antigen;

iii) T cells recognizing a specific antigen; and the combination of (a) and (b),

iv) T cell activity initiated in response to a particular antigen.

The specific antigen can then be administered to an individual identified as having a pre-existing immune response such that the antigen is introduced into the cancer, thereby treating the cancer.

In any of the methods provided in the present disclosure, other agents may be used (i.e., administered) in combination with CMV antigens within the practice of the present invention to enhance immune regulation or recruitment. Such other agents include TLR agonists; intravenous immunoglobulin (IVIG); peptidoglycan isolated from a gram positive bacterium; lipoteichoic acid isolated from gram positive bacteria; lipoproteins isolated from gram positive bacteria; lipid arabinomannan isolated from mycobacteria, zymosan isolated from yeast cell walls; poly (a) -poly (uridylic acid); poly (IC); a lipopolysaccharide; monophosphoryl lipid a; flagellin; gadimod (Gardiquimod); imiquimod (Imiquimod); r848; an oligonucleotide containing a CpG motif, a CD40 agonist, and 23S ribosomal RNA. In preferred aspects of these methods, the TLR agonist is poly-IC.

Another aspect of the disclosure is a kit for testing an individual and recruiting a pre-existing immune response to cancer in the individual. The kit can comprise at least one CMV peptide antigen or nucleic acid encoding a peptide, a pharmaceutically acceptable carrier, a container, and a package insert or label indicating administration of the CMV peptide to reduce at least one symptom of cancer in the patient. These kits may further comprise means (means) for testing the antigenic response of the patient to CMV antigens. For example, the kit may include sterilized plastic articles for obtaining and testing whole blood samples and for in vitro testing of responses to CMV peptide antigens and/or detecting interferon-gamma (IFN- γ) by enzyme-linked immunosorbent assay (ELISA) to identify in vitro responses to such peptide antigens.

Examples

Chronic viral infections, such as human cytomegalovirus (hCMV), which are often well controlled by the host, often lead to the induction of more and more fully functional virus-specific T cells with increasing age. Using a mouse mCMV model that mimics key aspects of the human immune response to hCMV, the present inventors developed methods and agents that attract these antiviral T cells to tumors, subsequently kill tumor cells and induce potent epitope spreading towards tumor neoantigens that leads to an adaptive immune response that confers long-term control of tumor growth and provides protection from re-attack by homologous tumor cells.

Example 1

Murine cytomegalovirus infection induces cytokine responses against the mCMV peptide library

C57Bl/6 mice were infected with 1x10^4pfu murine cytomegalovirus (mCMV). Blood samples were collected on day 12 post infection. Restimulation of blood leukocytes with a pool of selected immunogenic peptides from m38, m45, m57, m122, 1m39, m141 and m164 mCMV proteins. CD8+ T cells were evaluated for IFN-gamma, TNF-alpha and IL-2 cytokine production by intracellular cytokine staining and analyzed by Fluorescence Activated Cell Sorting (FACS) (FIG. 1A). Blood samples were collected two months after infection. Swelling (m122) and non-swelling (m45) specific CD8+ T cells were detected by FACS using MHC-I tetramer staining. Memory CD8+ T cell responses were mapped against mCMV. Spleens were collected six months after infection. Evaluation of restriction at m38, m45, m122 MHC-I and m139 by intracellular cytokine staining_560-574IFN-gamma production by CD8+ and CD4+ T cells following in vitro stimulation with MHC-II restricted mCMV peptide (FIG. 1B).

Example 2

Intratumoral transduction of solid tumors with HPV Psv expressing the mCMV antigen

C57Bl/6 mice were infected with 1x10^4pfu murine cytomegalovirus (mCMV). Six months post-infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins (injection protocol, FIG. 2A). Tumor growth was measured using electronic calipers. On days 13 and 15 post tumor injection, HPV16 Psv expressing m122 and m45 (FIG. 2B) or HPV Psv expressing Red Fluorescent Protein (RFP) (FIG. 2C) was injected intratumorally (every PsV 10^8 infectious units).

Example 3

Intratumoral transduction of solid tumors with mCMV antigen in combination with poly (I: C)

C57Bl/6 mice were infected with 1x10^4pfu murine cytomegalovirus (mCMV). Four months post-infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins (FIG. 3A). Intratumoral injection of tumors with HPV16 expressing m122, m38 and m45 or control RFP on days 11 and 13, HPV45 expressing m122, m38 and m45 or control RFP on days 16 and 18, and HPV58 expressing m122, m38 and m45 or control RFP (10 ^8 infectious units per PsV 10) on days 21 and 23, with or without poly (I: C) (30 μ g) (PIC). Tumor growth was measured using electronic calipers (fig. 3B-3E). These tumor volume/growth data indicate that intratumoral transduction of solid tumors with HPV Psv expressing the mCMV antigen slowed tumor growth, and that co-administration with poly (I: C) further slowed tumor growth (compare fig. 3B and 3D; and compare fig. 3C and 3E). Infiltration of tumors by E7 (fig. 3F), m45 and m122 (fig. 3G) specific CD8+ T cells was analyzed by MHC-I tetramer staining and FACS. These data indicate that tumor infiltration of CD8+ T cells is significantly enhanced when these CMV antigens are administered in combination with poly (IC).

Example 4

Intratumoral injection of mCMV MHC-I restricted peptides conferring increased survival

C57Bl/6 mice were infected with 1x10^4pfu murine cytomegalovirus (mCMV). Four months post-infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins (FIG. 3A). Tumors were injected intratumorally on days 11, 13, 16, 18, 21 and 23 with selected m38, m45 and m122 peptides (1 μ g each), with or without poly (I: C) (30 μ g), and either saline as a control or poly (I: C) alone. Animal deaths were recorded (fig. 4A) and tumor growth was measured using electronic calipers (fig. 4B). These data indicate that intratumoral injection of mCMV MHC-I restricted peptides delays tumor growth and confers increased survival.

Example 5

Intratumoral injection of mCMV MHC-I restricted peptides to delay tumor growth

C57Bl/6 mice were infected with 1x10^4pfu murine cytomegalovirus (mCMV). Four months post-infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. The tumors were injected intratumorally with progressively lower doses (1. mu.g, 0.1. mu.g and 0.01. mu.g) of selected m38, m45 and m122 peptides with or without poly (I: C) (30. mu.g) on days 11, 13, 16, 18, 21 and 23, and either as control saline or poly (I: C) alone. Tumor growth was measured using electronic calipers (fig. 5). These data indicate that intratumoral injection of mCMV MHC-I restricted peptide delays tumor growth.

Example 6

Combination of mCMV MHC-I and MHC-II restricted peptides for delaying tumor growth

C57Bl/6 mice were infected with 2.5x10^5 mCMV. Four months post-infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. Tumors were injected intratumorally 6 times with MHC-I restricted selected m38, m45, and m122 peptides and/or MHC-II restricted m139 selected peptides or saline from day 12 to day 28. All peptides were injected with poly (I: C) (30. mu.g). Groups were injected 6 times with MHC-I, or 6 times with MHC-II peptides, or 6 injections of both MHCI and MHCII peptides together, or 3 MHC-I peptides in sequence followed by 3 MHC-II peptides, or 3 MHC-II peptides followed by 3 MHC-I peptides. Tumor growth was measured using electronic calipers (fig. 6A and 6B). These data indicate that intratumoral injection of a combination of mCMV MHC-I and MHC-II restricted peptides delayed tumor growth. E7, m45, m122 specific CD8+ T cell responses in blood were also analyzed by FACS using MHC-I tetramers against each peptide (fig. 6C). These data indicate that intratumoral inoculation with mCMV CD4 followed by CD8 epitope preferentially induced anti-tumor immunity.

Example 7

Complete removal of primary tumors confers long-term tumor protection

S.c. injections of protected C57Bl/6 mice that survived primary tumor challenge as described in example 6 were performed with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins on the opposite side of the primary challenge. As a control for tumor capture (tumor take), young (12 weeks of age) and age-matched (10 months of age) mice were challenged with TC-1 tumor cells. Tumor growth was measured using electronic calipers (fig. 7). These data indicate that complete clearance of the primary tumor confers long-term protection against secondary tumor challenge.

Example 8

Intratumoral injection of MCMV to alter the tumor immune microenvironment

Two days after the end of the last intratumoral treatment, the effect of intratumoral injection of mCMV MHC-I and MHC-II restriction peptides on the tumor immune microenvironment with or without poly IC was analyzed in RNA samples for immune gene expression using a nanowire cancer immunology gene set (nCounter). The results are summarized by the change in score for each analyzed gene set. An overall score for differential expression of the gene set was performed relative to the saline-treated groups (n-4 per group). The microenvironment characteristics evaluated included: b cell function, interleukins, TNF superfamily, antigen processing, MHC, adaptation, transporter function, adhesion, NK cell function, T cell function, CD molecules, leukocyte function, complement pathway, microglial (microroglial) function, humoral, TLR, inflammation, dendritic cell function, interferon, innate, macrophage function, chemokines and receptors, aging, apoptosis, cytokines and receptors, cancer progression, essential cell function, cell cycle and pathogen response.

Example 9

mCMV infection induces dilatant CD8 in C57BL/6 mice⁺T cell response

C57Bl/6 mice were infected with 5x10^3pfu murine cytomegalovirus (mCMV). Blood samples were collected 1 or 5 months after infection. Both swelling (IE3) and non-swelling (m45) specific CD8+ T cells were detected by FACS using MHC-I tetramer staining. As shown in figure 8, mCMV infection induced a clear effector and memory CD8+ T cell response.

Example 10

mCMV infection induced potent CD8 in C57BL/6 mice⁺And CD4⁺T cell response

C57Bl/6 mice were infected with 5x10^3pfu murine cytomegalovirus (mCMV). Blood samples were collected on day 12 post infection. Splenocytes were restimulated with the indicated peptides and blood cells were restimulated with a library of immunogenic peptides with selections from m38, m45, m57, m122, m139, m141, and m164 mCMV proteins. IFN-gamma, TNF-alpha and IL-2 cytokine production by CD4+ and CD8+ T cells was assessed by intracellular cytokine staining and analyzed by FACS (FIGS. 9A, 9B). These results indicate that murine cytomegalovirus infection induces a large cytokine response.

Example 11

Tissue distribution of mCMV-specific CD8+ T cells

The distribution of mCMV-specific CD8+ T cells in tumor-bearing mice was studied. Using 5x10^3

mCMV infects C57Bl/6 mice. The experimental schedule is shown in fig. 10A. Four months post-infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. Lymph nodes, spleen, salivary glands and tumor tissue were collected and expansion (IE 3; fig. 10B) and non-expansion (m 45; fig. 10C) specific CD8+ T cells were detected by FACS using MHC-I tetramer staining. Expression of resident memory T cell markers was assessed using CD69 and CD103 antibodies. These results indicate that TC1 tumors were infiltrated by mCMV-specific CD8+ T cells.

Example 12

Gene expression analysis of tumor microenvironment

The expression of genes in tumor cells in a mouse model after intratumoral treatment with (4 animals per group) was studied: brine; poly I C (PIC) (50. mu.g); mCMV m139 peptide (MHC-II restricted/CD 4) (CD4) (3 μ g); mCMV m38, m122, m45 peptide (MHC-I restricted/CD 8) (CD8) (1. mu.g each); mCMV m139+ poly I: C (PIC CD4) (3. mu.g each); mCMMm 38, m122, m45 peptide (MHC-I restricted/CD 8) + Poly I: C (PIC CD8) (1. mu.g each). Tumors were treated 3 times at weeks 11, 13, and 16 after subcutaneous placement of TC1 tumor cells. The protocol schedule is shown in figure 11A. After treatment and harvesting of tumors, tumor RNA was extracted using qiatube. Tumor cell gene expression was analyzed using a nanowire oncogene set (NS _ MM _ CANCERIMM _ C3400) in the form of gene transcripts measuring 770 genes in a tumor PanCancer immune profiling Panel (PanCancer immune profiling Panel). Briefly, normalized data are presented as heatmaps of gene set expression in specific biological processes (adaptive immunity, antigen processing, T cell function, dendritic cell function, NK cell function, interferon, TNF superfamily genes); volcano plots of gene expression changes relative to saline treatment were constructed (the plots show changes in the treatment groups (expressed as fold increase or decrease) relative to control treatment (saline) and are statistically significant); cell infiltration quantification algorithms (CD45, cytotoxic CD8, CD4 Th1, NK cells and dendritic cells) were applied. The results show that the global significance scores varied most in MHC-I restricted/CD 8 and MHC-I restricted/CD 8+ poly (I: C) treated animals.

Profile analysis of the immune genes in the whole tumor RNA after intratumoral treatment showed significant upregulation of immune genes in three groups:

1) mCMV m139 peptide: MHC-II restriction/CD 4-3mg (230 genes up-regulated, 4 genes down-regulated);

2) mCMV m38, IE3, m45 peptide: MHC-I restriction/CD 8-1mg (359 genes up-regulated and 43 genes down-regulated);

3) mCMV m38, IE3, m45 peptide: MHC-I restriction/CD 8+ poly (I: C) (309 genes up-regulated, 49 genes down-regulated).

After intratumoral treatment, leukocyte infiltration into the tumor was also analyzed. FIGS. 11B-11F show tumor infiltration by different leukocytes. These data indicate that intratumoral injection of the CD8 mCMV epitope (with or without poly (I: C)) induces recruitment of T cells and non-T cells (NK) in tumors; and intratumoral injection of the CD4 mCMV epitope with poly (I: C) induces recruitment of T cells and non-T cells (NK) in the tumor; and intra-tumoral injection with a poly (I: C) of the CD8 or CD4 epitope induces recruitment of dendritic cells in the tumor.

Example 13

Intratumoral injection of the mCMV CD8 epitope to delay tumor growth

C57Bl/6 mice were infected with 5x10^3pfu murine cytomegalovirus (mCMV). Four months post-infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. Tumor growth was measured using electronic calipers. Tumors were injected intratumorally with selected MHC-I restricted m38, m45, and m122 peptides (0.01, 0.1, or 1 μ g each) with or without poly (I: C) (30 μ g) on days 11, 13, 16, 18, 21, and 23, and saline or poly (I: C) alone as controls. Figures 12A and 12B show that intratumoral injection of mCMV MHC-I restricted peptide delayed tumor growth and that poly (I: C) coinjection improved tumor control.

Example 14

Protection from TC1 and MC38 tumor challenge by intratumoral injection of mCMV MHC-I and/or MHC-II peptides with poly (I: C)

C57Bl/6 mice were infected with 5x10^3 mCMV. Four months post-infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. Tumor growth and survival were monitored. Tumors were injected intratumorally 6 times with MHC-I restricted selected m38, m45 and m122 peptides and/or MHC-II restricted m139 selected peptides with or without poly (I: C) (30 μ g) from day 12 to day 28, and saline or poly (I: C) alone as control. Groups were injected 6 times with MHC-I, or 6 times with MHC-II peptide, or 6 times together with MHCI and MHCII peptide, or 3 times in sequence with MHC-I peptide followed by 3 times with MHC-II peptide, or 3 times with MHC-II peptide followed by 3 times with MHC-I peptide. Figure 13A shows that intratumoral injection of a combination of mCMV MHC-I and MHC-II restricted peptides delayed tumor growth, and figure 13B shows that intratumoral inoculation with CD4(MHC-II), followed by CD8(MHC-I) mCMV epitope in sequence promoted long-term survival.

Example 15

E7 tetramer positive CD8 in blood after treatment⁺T cell response

C57Bl/6 mice were infected with 5x10^3 mCMV. Four months post-infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. Tumor size was measured using electronic calipers. Tumors were injected intratumorally 6 times with MHC-I restricted selected m38, m45 and m122 peptides and/or MHC-II restricted m139 selected peptides with or without poly (I: C) (30 μ g) from day 12 to day 28, and saline or poly (I: C) alone as control. All peptides were injected with poly (I: C) (30. mu.g). Groups were injected 6 times with MHC-I, or 6 times with MHC-II peptide, or 6 times together with MHCI and MHCII peptide, or 3 times in sequence with MHC-I peptide followed by 3 times with MHC-II peptide, or 3 times with MHC-II peptide followed by 3 times with MHC-I peptide. E7, m45, m122 specific CD8+ T cell responses in blood were analyzed by FACS using MHC-I tetramers against each peptide. Figure 14 shows intratumoral inoculation with mCMV CD4 followed by CD8 epitope in order to preferentially induce anti-tumor immunity.

Example 16

Long term protection against secondary tumor challenge

S.c. injections of protected C57Bl/6 mice that survived primary tumor challenge as described above were injected with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins on the opposite side of the primary challenge. Tumor growth was measured using electronic calipers. As a control for tumor capture, young (12 weeks of age) and age-matched (10 months of age) mice were challenged with TC-1 tumor cells. Figure 15 shows that complete clearance of the primary tumor conferred long-term protection against secondary tumor challenge.

Example 17

Protection from poly (I: C) by intratumoral injection of mCMV MHC-I and MHC-II peptides

MC38 tumor challenge

C57Bl/6 mice were infected with 5x10^3 mCMV. Four months post infection, mice were injected s.c. with 5x10^5MC 38 tumor cells from mouse colon adenocarcinomas that exhibit hyper-mutation and microsatellite instability. Tumor growth was monitored. Tumors were injected intratumorally 6 times with MHC-I restricted selected m38, m45 and m122 peptides and MHC-II restricted m139 selected peptides with poly (I: C) (30 μ g) from day 12 to day 28, or MHC-II restricted m139 selected peptides alone with poly (I: C) (30 μ g) and saline alone as control. Figure 16 shows that complete clearance of the primary tumor conferred long-term protection against secondary tumor challenge. FIG. 16 shows that intratumoral injection of a combination of mCMMHC-I and MHC-II restricted peptides delayed tumor growth and resulted in tumor clearance.

The studies described in examples 1-17 indicate that both non-swelling and swelling mCMV specific T cells infiltrate tumors during potential mCMV infection and redirect established antiviral T cells into solid tumors leading to tumor regression, a dramatic change in the tumor immune microenvironment. The data also show that redirecting established antiviral CD4+ T cells into solid tumors promotes epitope spreading to tumor associated antigens and complete tumor clearance. Thus, these methods provide a widely applicable "antigen agnostic" tumor therapy based on pre-existing antiviral T cells. HPV L1 and L2 particles showed strong tropism for numerous tumor cells, but did not bind or infect intact epithelium. Thus, HPV PsV or VLP can be used to genetically or directly as a carrier to direct anti-tumor agents to tumor cells.

While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to fall within the scope of the claims.

Sequence listing

<110> United states of America, represented by the health and human service Institution

Schiller, John T.

Cuburu, Nicolas

Lowy, Douglas R.

<120> novel cancer treatment using pre-existing microbial immunity

<130>6137NCI-56-PCT

<140> not yet allocated

<141>2018-11-06

<150>62/582,097

<151>2017-11-06

<160>67

<170>PatentIn version 3.5

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Claims (29)

1. A method of treating cancer in an individual comprising recruiting a preexisting immune response to a site of the cancer, thereby treating the cancer.

2. The method of claim 1, wherein the pre-existing immune response is a naturally occurring pre-existing immune response.

3. The method of claim 1 or 2, wherein recruiting the pre-existing immune response to a cancer cell comprises introducing an antigen not expressed by a cancer cell into the cancer prior to initiation of treatment, wherein the antigen is recognized by one or more components of the pre-existing immune response.

4. The method of any one of the preceding claims, wherein the individual is confirmed to have a pre-existing immune response against the antigen prior to introducing the antigen into the tumor.

5. The method of claim 4, wherein the step of confirming the presence of the pre-existing immune response comprises identifying a T cell response to the antigen in a sample from the individual.

6. The method of any one of the preceding claims, wherein the step of introducing the antigen comprises injecting the antigen into the cancer.

7. The method of any one of the preceding claims, wherein the step of introducing the antigen comprises introducing a nucleic acid molecule encoding the antigen into the cancer.

8. The method of claim 7, wherein the nucleic acid molecule is DNA.

9. The method of claim 7, wherein the nucleic acid molecule is RNA.

10. The method of claim 9, wherein the RNA is modified to make it more resistant to degradation.

11. The method of claim 7, wherein the nucleic acid molecule is introduced into the cancer by injection.

12. The method of claim 7, wherein the nucleic acid molecule is introduced into the cancer using a viral vector.

13. The method of claim 12, wherein the viral vector is introduced into the cancer using a pseudovirion.

14. The method of claim 13, wherein said pseudovirion is a papilloma virus pseudovirion.

15. The method of any one of claims 3-14, wherein the antigen is a viral antigen.

16. The method of any one of claims 3-14, wherein the antigen is a polypeptide comprising at least one epitope from a Cytomegalovirus (CMV) protein, and wherein the at least one epitope is recognized by one or more components of the pre-existing immune response.

17. The method of claim 16, wherein the one or more components are T cells.

18. The method of claim 16, wherein the CMV protein is selected from the group consisting of: pp50, pp65, pp150, IE-1, IE-2, gB, US2, US6, UL16 and UL 18.

19. The method of claim 16, wherein the polypeptide is an MHC I-restricted peptide of 9-15 amino acids.

20. The method of claim 16, wherein the polypeptide is an MHC II restricted peptide of at least 15 amino acids.

21. The method of claim 16, wherein the antigen comprises a sequence at least 90% identical to a sequence selected from the group consisting of seq id nos: 1-67 of SEQ ID NO.

22. The method of claim 16, wherein the antigen comprises a sequence selected from the group consisting of SEQ ID NOs 1-67.

23. The method of any one of claims 3-22, wherein recruitment of the pre-existing immune response alters the microenvironment of the cancer selected from the group consisting of B cell function, interleukins, TNF superfamily, antigen processing, MHC, adaptability, transporter function, adhesion, NK cell function, T cell function, CD molecules, leukocyte function, complement pathway, microglial function, humoral, TLR, inflammation, dendritic cell function, interferon, innate, macrophage function, chemokines and receptors, aging, apoptosis, cytokines and receptors, cancer progression, essential cell function, cell cycle, and pathogen response.

24. The method of any one of claims 3-23, wherein the antigen is administered in combination with an agent that enhances the immune response selected from the group consisting of a TLR agonist; an IL-1R8 cytokine antagonist; intravenous immunoglobulin (IVIG); peptidoglycan isolated from a gram positive bacterium; lipoteichoic acid isolated from gram positive bacteria; lipoproteins isolated from gram positive bacteria; lipid arabinomannan isolated from mycobacteria, zymosan isolated from yeast cell walls; poly (a) -poly (uridylic acid); poly (IC); a lipopolysaccharide; monophosphoryl lipid a; flagellin; gadimod (Gardiquimod); imiquimod (Imiquimod); r848; an oligonucleotide containing a CpG motif, a CD40 agonist, and 23S ribosomal RNA.

25. The method of any one of claims 3-23, wherein the antigen is administered in combination with poly-IC.

26. The method of any one of the preceding claims, wherein the cancer is a solid tumor.

27. The method of any one of the preceding claims, wherein the cancer is a hematological cancer.

28. A kit for recruiting a pre-existing immune response to cancer in an individual, comprising at least one CMV peptide antigen or nucleic acid encoding the peptide, a pharmaceutically acceptable carrier, a container, and a package insert or label describing administration of the CMV peptide to reduce cancer in a patient.

29. A kit for testing a patient and recruiting a pre-existing immune response to a site of cancer in the patient.

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